Biological Forum An International Journal 7(1): 736-740(2015) ISSN No. (Print): 0975-1130 ISSN No. (Online): 2249-3239 The Effect of Different Salinities on Density of Spirulina plaetensis under Laboratory Conditions Esmaeil Kouhgardi *, Elham Moazami *, Reza Ghorbani-Vaghei ** and Tirdad Maghsoudloo * *Department of Fisheries, Bushehr Branch, Islamic Azad University, Alishahr, Bushehr, IRAN. **Iranian Fisheries Research Organization- Shrimp Research Center. IRAN (Corresponding author: Esmaeil Kouhgardi) (Received 17 January, 2015, Accepted 15 March, 2015) (Published by Research Trend, Website: www.researchtrend.net) ABSTRACT: This research conducted with five treatments of salinity of 15, 20, 25, 30 and 35 ppt on culture of S. plaetensis. Comparing the average of density of the species under treatments revealed that the least density is at salinity of 35 ppt. A significance difference was at the level of 5% comparing to other treatments. The most density was obtained in treatment of 15 ppt. The most cellular density was for 15, 20 and 25 ppt and salinities of 30 and 35 ppt rated 4 th and fifth respectively. Relying on above statements, one may come up with this conclusion that cellular density of S. plaetensis is less in lower salinities than that of higher salinities. Upon raise of degree of salinity, growth of the species shall decrease. In other words, in its natural habitation, this species tolerate higher salinity. However, the nature of the species is more compatible with lower salinity. Key Words: Spirulina plaetensis, Cellular Density, Salinity, Nature of Species INTRODUCTION Microalgae are the species that are capable of producing valuable metabolites such as pigments, proteins and vitamins for edible additives, medical products and other goals. From among these microalgae, one may name Spirulina plaetensis which is one of the most prominent species of alga that has been studied in biotechnology. Thanks to easiness of growth of species, its drying process, and due to its high protein ingredient up to 70%, specific pigment especially the blue one named Phicociyanin, fatty acids such as Linolenic acid, B 12 vitamin and minerals, the species has received great attention all over the world [3]. Further to success of Spirulina in aquatic farming and poultry feeding, ph and temperature are among the key elements involved in farming the said kind of alga in large scale. The optimized temperature for farming Spirulina is 35-38 degrees centigrade. Further to the required ph for S.plaetensis is given as 9.1-9.5 that effectively prevents contamination of most algae while farming. Thus, high quantities of Sodium Bicarbonate must always exist in the medium[3]. The name of Spirulina has been taken from its spiral form. Further to its high nutrients, Spirulina is every effective in response to safety system and protection against radiation [4]. As a foodstuff that provides health, Spirulina plays a prominent role in prevention and improvement of symptoms of diseases. Hormones, antibiotics and other chemicals that have positive effect also enjoy destructive environmental effects, high price and development of resistant bacteria. Thus, tendency toward safety stimulants have promoted. Use of Spirulina instead of antibiotics causes decrease of water contamination, decrease of medical costs and promotion of efficiency of farming systems [8]. Spirulina is introduced as a diet and its effect on growth, survival, safety and different tissues of animals have been studied [2]. Various studies have been conducted in the field of using Spirulina as feed for animals, human and aquatics [1]. For example, using Cyprinus carpio as diet, different species of Spirulina has been replaced fish powder as a source of protein. MATERIALS AND METHODS The required means consist of 15 bottles of 2 liters per each, colorized water (that has been blown) with salinity of 40 ppt, two florescent lamps, blowing pump, Spirulina Stock, and Conway medium. For producing the required salinities, it is required to mix the drinkable water with salt water with a salinity of 40 ppt, the water of the required salinities (15, 20, 25, 30 and 35 ppt) has been obtained.
Kouhgardi, Moazami, Ghorbani-Vaghei and Maghsoudloo 737 Five treatments with three treatments have been considered. Blowing has been done by using an air pump. Light with an intensity of 5000 Lux and temperature of about 28 degrees centigrade has been constant. Spirulina has been supplied in stock form through Alga Culture Laboratory of Bushehr in southern parts of Iran. Conway medium had been prepared before and then, it entered the farming environment after its temperature has equalized to that of the environment for about 1.5 milliliter and then, after being homogenized, Spirulina stock of about 15 milliliters was added to the said environment. One cc of water of the farming environment was put on microscope every day and cellular density of Spirulina has been numerated and increasing or decreasing process of its growth has been studied. The specific slide consists of 1000 equalized cells. Four cells have been selected at random. Number of the cells placed on these four parts of the slide has been numerated and corresponding average has been calculated accordingly. String Spirulina alga consists of some small and large spirals. Each spiral consists of nine cells. Multiplying number of spirals per nine cells, number of cells of the alga is obtained. The following formula was used to obtain number of cellular density in one milliliter of water: Spirulina in one milliliter of solution = X*9*20*103/4 cellular density X = number of spirals of Spirulina spiral alga 9 = number of cells of each spiral 20 = Dilution percentage 10 3 = Number of cells of the numerate slide 4 = Number of numerated cells of the slide RESULTS The results of numerating cellular density of S. plaetensis in salinities of 15, 20, 25 and 35 during a period of 14 days and average of cellular density numerated, are given in Tables 1 and 2 as well as in Fig. 1. Corresponding results of data analysis for density of the species under different treatments of salinity during a period of 14 days of farming revealed that these treatments had no significant difference in the second day. However, on the 1 st, 3 rd, 4 th, 5 th, 7 th, 9 th, 10 th, 11 th, 12 th and 13 th day with a probability of 95% is significant. On the 6 th, 8 th, and 14 th, with a probability of 99%, it is significant as well. Similar small letters on the right side designates significant indifference (P>0.05) and dissimilar letters designates significant difference in columnar form (P<0.05). Table 1: Analysis of cellular density of S. plaetensis affected by different treatments of salinity. Mean Square Days of Culture Source of 1 2 3 4 5 df change Treatment 4 11089.12* 21090 ns 586710* 364627.5* 306360* Error 10 5835.6 117420 341820 262950 264720 6 7 8 9 10 Treatment 4 3751080.9** 362275.68 * 1555034.23 ** 528530.22* 695267.1* Error 10 2460419.16 326757.12 729660 30980122 583492.3 11 12 13 14 Treatment 4 908405.4* 655635.6* 504447* 1747347.48** Error 10 801354 369416.8 106497.12 1354948.56 No significant difference: ns *Significant difference at level of 5% **Significant difference at level of 1%
Kouhgardi, Moazami, Ghorbani-Vaghei and Maghsoudloo 738 Table 2: Comparing the average of cellular density of S. plaetensis in different treatments of salinity. Cellular Density Days of Culture Salinity (ppt) 1 2 3 4 5 15 58.5± 5.3 c 760± 12ª 1010± 21 ª 1100± 21ª 1070± 14ª 20 74± 4 c 670± 11ª 810± 12 b 1025± 21ª 1090± 9ª 25 89± 5.5 c 620± 10ª 1340± 13ª 950± 13ª 820± 11ª 30 106.5± 6 b 590± 9ª 360± 21 c 955± 14ª 950± 12ª 35 212± 7ª 780± 11ª 290± 13 c 240± 11 b 310± 13 b 6 7 8 9 10 15 1030± 23 b 680± 20 b 1010± 23 b 1150± 21ª 1200± 32ª 20 2880± 32ª 890± 12 b 540± 11 c 490± 11 b 590± 21 b 25 990± 15 b 1030± 13ª 1950± 36ª 475.33± 6.31 b 960± 12ª b 30 220± 11 c 740± 14 b 710± 14 c 320.02± 14 b 320.02± 14 c 35 70.02± 5 d 120.04± 15 c 0.06± 0.01 d 0.06± 0.01 c 0.06± 0.01 d 11 12 13 14 15 1340± 24ª 980± 21ª 1020± 21ª 1870± 25 ª 20 1040± 25 b 960± 31 b 960± 31 b 980± 23 b 25 690± 23 b 510± 15 b 510± 15 b 270.02± 32 c 30 250.04± 15 c 130.04± 14 c 120.04± 24 c 240.04± 31 c 35 0.06± 0.01 d 0.06± c 0.01 0.06± 0.01 d 0.06± 0.01 d Numbers by a factor of 1.5*10 3 is calculated 3500 3000 2500 Cell number 2000 1500 1000 500 15 ppt 20 ppt 25 ppt 30 ppt 35 ppt 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Culture days Fig. 1. Growth of Spirulina affected by different salinities during a fourteen-day period of culture.
Comparing the average of cellular density of Spirulina affected by different treatments during a fourteen day period of culture Table of comparing density of the alga in the first day revealed that the least density of alga was for treatment 15 and 20 ppt. It showed a significant difference at the level of 5% with other treatments. The most density of alga was obtained in treatment 35 ppt. In the second day it showed a significant difference with other treatments at level of 5%. The most density of the alga was in treatment 35 ppt. In the second day, it has been revealed that there was no significant difference either. In other days except for the sixth day, the most density of alga was in treatment 15 ppt and revealed a significant difference with other treatments at levels of 5% and 1% (7 th and 14 th days) respectively and the least density was for density of alga in treatment 35 ppt and Kouhgardi, Moazami, Ghorbani-Vaghei and Maghsoudloo 739 the third day of treatment 25 and the sixth day of treatment 20 ppt showed the most density of alga and a significant difference with other treatments was revealed at level of 1%. The least density of alga was obtained in treatment 35 ppt. Corresponding results to data analysis of cellular density affected by different treatments of salinity revealed that these treatments have a significant difference at level of 5%. Comparing the average of cellular density of S. plaetensis affected by different treatments of salinity showed that the least cellular density is in salinity of 35 ppt (144.46+/-21.34), revealing a significant difference with other treatments at level of 5%. The most cellular density was obtained at treatment of 15 ppt (1019.89+/-85.65). On the whole, it may be concluded that cellular density of the Spirulina at lower salinities is more than higher salinities. Table 3: Analysis of variable of cellular density of S. plaetensis affected by different treatments of salinity during culture period. Resources of changes df Mean of squares Treatment 4 18901.12* Error 10 5385.6 No significant difference: ns *Significant difference at level of 5% **Significant difference at level of 1% Table 4: Comparing cellular density of S. plaetensis after applying different treatments of salinity during culture period. DISCUSSION Salinity (ppt) Cellular Density 15 1019.89±36.05 a 20 912.07± 25.54 ab 25 808.88± 41.2 b 30 429.4± 25.7 c 35 144.94± 16.8 d Numbers by a factor of 1.5*10 3 is calculated Investigations carried out on growth process of species of S. plaetensis affected by salinity of 15 ppt revealed that cellular density of this species has had an increasing trend from the beginning through the end of process except for the 7 th and 12 th day during which it showed a slight decrease. However, growth diagram of the said species affected by salinity of 35 ppt revealed that cellular density had a slight increase in the second day and followed a decreasingly trend until the 8 th day when it reaches zero. Thus, one may come up with this conclusion that upon increase of salinity, cellular density of the Spirulina has been decreased and at lower salinities, it shall have an increasing and more proper growth accordingly. Further to physical elements (light, temperature, blowing and the ones), S. plaetensis needs a medium for fulfillment of its vital activities. The medium or the nutrition consists of macro-element and micro-element [9] and in this research, for nutrition supply of S. plaetensis, Conway medium was used since it had nitrate, phosphate, potassium and ferrous as well as vitamin solutions in its formula.
Kouhgardi, Moazami, Ghorbani-Vaghei and Maghsoudloo 740 The results obtained from the study of growth of S.plaetensis in four mediums of Conway, Gilard, N 8 and TMRL at salinity of 15 ppt, the most growth was obtained at Conway medium [7]. In this research, since the medium (Conway) was constant, the most growth was obtained at salinity of 15 ppt. Temperature is one of the most important factors affecting the biochemical compound of the alga. Temperature above 35 degrees centigrade is fatal for some species. However, for others like S.plaetensis, the most proper temperature for farming is given as 35-38 degrees centigrade. This problem was mentioned in the research by Goksan [3] which studied the growth of S.plaetensis in different culture systems under greenhouse conditions. However, higher temperature than 35 degrees centigrade is really dangerous for Spirulina [5]. Concerning the fact that the said test was conducted in winter and low temperature was one of the main elements that limited growth in winter, temperature higher than 28 degrees centigrade was maintained and this caused proper growth of S. plaetensis at desirable salinity. The water used for culture of Spirulina must be clean, filtrated and free from any microbes and contamination and enjoys all necessary low-consumed elements and the required compounds. The drinkable water may be used as well. It enjoys enough quantity of calcium. However, if the quantity of calcium exceeds the said amount, it causes slugging and sedimentation of the same and this shall bring about some problems. The limit of light intensity for culture of microalgae is given as 1000-10000 Lux. Its optimum amount is given as 2500-5000 [6]. The light intensity used in this research is given as 5000 Lux. The light period during 24 hours a day is not recommended because during darkness period, synthesis of protein, respiration and other internal reactions are conducted [5]. Concerning the fact that in this test, only cellular density of Spirulina alga and its decreasing and increasing process of growth was considered, the said test was conducted during a light period of 24 hours of brightness. Culture and harvesting Spirulina alga under laboratory conditions revealed that on the whole from applied treatments of salinity of 1-20% (1, 4, 10, 15 and 20%), upon increase of salt concentration, growth has decreased. In this research which was carried out under similar conditions, growth has decreased at higher degrees of salinity [10]. The most growth was observed at salinity degree of 15 ppt. Based on the results, the most cellular density was at salinity of 15 ppt and then, the salinity of 20 ppt obtained the second place. The salinity of 25 ppt obtained the third place and the salinities of 30 ppt and 35 ppt obtained the 4 th and 5 th places respectively. Relying on above statements, it may be concluded that upon increase of degree of salinity, cellular growth of Spirulina is decreased. REFERENCES Aslianti, Y. (1988). Experiment on the mass production of dried Spirulina for fish and shrimp food. Journal penelitian Budidaya Pantai Maros. 10(3): 9-16. Belay, A. (2002). The potential Application of Spirulina (Arthrospira) as a nutrition and therapeutic supplement in health management. Journal of the American Nutraceutical Association. 5: 27-48. Goksan, T., Zekeriyaoglu, A. & AK, I. (2006). The growth of Spirulina platensis in different culture systems under greenhouse conditions. Turkish Journal of Biology. 31: 47-52. Jaime-Ceballos, B., Hernández-Llamas, A., García, T., Perez-Jar, L. & Villareal, H. (2006). Substitution of Chaetoceros mulleri by Spirulina platensis Meal in Diets for Litopenaeus schmitti Larvae. Journal of Aquaculture. 266: 215-220. Jourdan, F. (2001). Manual of small Scale Spirulina culture, Antenna Technologies, p.1-16. Lavens P. & Sargeloos P. (1996). Manual on production and use of live food for aquaculture. FAO Technical Report, pp. 295. Purzard, SH. & Ghaeni M. (2011). Evaluation of Spirulina platensis growth in the different mediums. In: Proceedings of National Conference on Aquatic, Iran, 1: 35-41. Sakai, M. (1999). Current research status of fish immune stimu land. Journal of Aquaculture. 172: 92-63. Salavatian, M., Azari Takami, Gh., Vahabzadehrodsari H. & Rajabinezhad R. (2007). Evaluation of growth and biomass of Nannochloropsis oculata algae. Iranian Journal of Marine Sciences. 5(1, 2): 43-53. Soltani, N. & Baftehchi, L. (1996). Culture plan, harvesting of Spirulina algae in the Laboratory scale. M.Sc. Thesis of Shahid Beheshti University, Iran: pp.140-170.